There are two specific goals of this project. The first is to characterize prothrombin activation as it occurs during in vitro blood clotting. We will study activation of both Factors ahd platelets and how subsequent prothrombin activation is influenced by these reactions. Human Factor V will be purified from fresh plasma. Thrombin-activated Factor Va (Factor Va) will be added to blood and its effect on prothrombin activation during in vitro clotting measured by our radioimmunoassay for thrombin. We will also make specific antibodies against Factor Va. Those antibodies which cross-react with Factor V will be removed by affinity chromatography. We will develop a radioimmunoassay for Factor Va using the anti-Factor Va antibody. The RIA will be used to study activation of Factor V during blood clotting. We will also determine the effect of the antibodies on thrombin formation. To determine the role of platelets in prothrombin activation, the effects of selectively activating platelets or varying the platelet concentration will be studied. To determine the role of platelet Factor V, platelets from Factor V-deficient patients or normal controls will be added to normal plasma and the effect on thrombin formation determined. These experiments will provide new information on the normal physiology of blood clotting. The second goal of this project is to further characterize the covalent binding of thrombin to platelets and endothelial cells. To obtain information on the functional inportance of the covalent binding of thrombin to endothelial cells, we will determine the relationship of binding to the inhibitory effect of thrombin on the plasminogena activity of endothelium. We will also determine whether binding of (125I) Factor Xa cultured bovine aortic endothelial cells is similar to thrombin binding to these cells. Covalent binding of activated coagulation factors to endothelium may serve as a mechanism for removing them from the circulation. Thrombin binding to endothelial cells will be localized using transmission electron microscopy and anti-thrombin IgG adsorbed to colloidal gold. We will also purify these covalent binding sites for thrombin from conditioned culture media from endothelial cells, and from the supernatant of platelets stimulated to undergo the release reaction. These studies should increase our understanding of the interaction between platelets, blood vessels, and clotting factors, and as a result, clarify the importance of these reactions in clotting.